The high prevalence of obesity in the US is a major public health concern, as overweight and obese individuals are at increased risk for many chronic diseases. Obesity stems from an imbalance between total caloric consumption and total energy expenditure (TEE), although the causes of this imbalance remain debated. Accurate measurements of TEE therefore play a pivotal role in understanding and ultimately reversing this epidemic.
TEE can be measured using direct (measurement of heat production) or indirect (measurement of respiratory gas exchange) calorimetry, but neither of these approaches are practical for measuring TEE in free living subjects. The gold standard for measuring TEE in free-living individuals is the doubly labeled water (DLW) method, which is based on the principle that the oxygen in body water is in complete isotopic equilibrium with the oxygen in dissolved respiratory carbon dioxide due to the action of carbonic anhydrase. The consequence of this exchange is that an isotopic label of oxygen introduced into body water is eliminated by the combined flux of body water and the exhaled carbon dioxide. Lifson and colleagues reasoned that, since hydrogen is found only in water and not in carbon dioxide, the elimination of a hydrogen isotope would be affected solely by the flux of body water. Thus the difference in the rates of isotope elimination of simultaneously administered oxygen and hydrogen labels is a measure of CO2 production. Review of the doubly labeled water technique, validation of its assumptions, and equations for calculating metabolic CO2 production from the isotopic measurements may be found, e.g., in D. A. Schoeller, “Measurement of Energy Expenditure in Free-Living Humans by Using Doubly Labeled Water”, Journal of Nutrition 118, pages 1278-89 (1988); J. R. Speakman, S. Nair, and M. I. Goran, “Revised equations for calculating CO2 production from doubly labeled water in humans”, American Journal of Physiology 264, pages E912-7 (1993); J. R. Speakman, “The history and theory of the doubly labeled water technique”, American Journal of Clinical Nutrition 68(suppl), pages 932S-938S (1998); and others, including references cited in the aforementioned papers.
Despite its usefulness, the DLW, method has some limitations. First, the test is expensive to perform due to the need for large quantities of H218O (approx. 0.25 gram per kilogram of a subject's fat-free mass) in addition to 2H2O. This expense is predominantly due to the cost of the 18O that is used to label subjects. High levels of 18O are required to distinguish the dose from fluctuating background isotope levels after 14-28 days of elimination; it currently costs $300-$400 for the 18O required to perform a DLW measurement on an adult subject (50-100 kg fat-free mass). Thus, widespread adoption of the DLW method has been limited by its high cost.
High levels of 18O tracer are needed to ensure that unknown fluctuations in the background isotope levels over time do not contribute excessively to measurement uncertainty. While the isotopic composition of atmospheric oxygen (O2) is itself essentially constant within the time frame of TEE testing, living test subjects also require regular food and water intake for good health, both of which are background sources of hydrogen and oxygen intakes. The isotopic composition of both natural water and various water-bearing foodstuffs vary according to factors such as local evaporation and precipitation rates at their source. Accordingly, daily variations in dietary and beverage intake by the test subjects contribute to the uncertainty in background isotope levels. This uncertainty in the background levels increases the isotope dose that must be administered and contributes to the uncertainty in the DLW measurements as compared to the reference calorimetry measurements of TEE in validation studies. Individual measurements are only precise to ±5%, so the method is currently most suitable for studies of groups rather than individual variation.
The proposed invention aims to address these two problems by significantly reducing the cost of the DLW method and improving the individual accuracy of the measurements.
E. R. T. Kerstel, R. Van Trigt, N. Dam, J. Reuss, H. A. J. Meijer, “Laser spectrometry applied to the simultaneous determination of the δ2H, δ17O, and δ18O isotope abundances in water”, IAEA-TECDOC-1247, pp. 7-13 (2001) describes application of infrared laser spectrometry to the simultaneous determination of the relative 2H/1H, 17O/16O, and 18O/16O isotope abundances in natural water. The method uses a narrow line width color center laser directed into gas cells equipped with multiple-pass reflection optics (for ≈20 m path length) to record the direct absorption spectrum of low-pressure gas-phase water samples in the 3 μm spectral region (ro-vibrational transitions around 3663 cm−1). The precision of the technique is shown to be 0.7‰ for δ2H and 0.5‰ for δ17O and δ18O, while the calibrated accuracy is about 3‰ and 1‰, respectively.
G. Lis, L. I. Wassenaar, and M. J. Hendry, “High-Precision Laser Spectroscopy D/H and 18O/16O Measurements of Microliter Natural Water Samples”, Anal. Chem. 80(1), pp. 287-293 (Jan. 1, 2008) describes use of off-axis integrated cavity output spectroscopy (OA-ICOS) for isotopic analysis (δD and δ18O) of water samples. A liquid autosampler injects from 0.2 to 1.0 μL of H2O into a pre-evacuated optical cavity via heated (70° C.) injection port to facilitate complete evaporation and vapor transfer through a tube. The highly reflective mirrors of the optical cavity extend the average optical path length to ˜3000 m allowing the use of infrared diode lasers operated at room temperature. The laser wavelength is tuned over the absorption spectrum of the isotopologues of interest of the injected H2O sample. Random instrumental drift was corrected by systematically spacing standard injections within the autorun and conducting linear interpolations. Potential intersample memory effects and mixing of water samples were overcome by using five sequential injections of each sample, discarding the first two injection results and accepting the mean of the final three injection results. Measurement accuracies of ±0.8‰ for δD and ±0.1‰ for δ18O were achieved.